This invention relates to a feeding tray for housing a nutrient medium utilized with a multiwell test apparatus suitable for promoting fluid interactions such as by growing cells in a nutrient liquid within a multiplicity of wells. More particularly, this invention relates to such a feeding tray of a multiwell test apparatus which permits adding or removing liquid from the feeding tray of the multiwell test plate without disturbing a material in the test plate such as cells within the wells.
At the present time, multiwell test apparatus for testing samples include a multiwell filter plate, a feeding tray, a multiwell receiver plate and a lid. The wells of the filter plate are formed of hollow members, typically tubular in shape although rectangular, square, triangular and hexagonal shapes have been used, with two open ends to one of which, typically the bottom one, is attached a membrane such as a microporous membrane. The members can be inserted into a feeding tray containing a nutrient medium so that cells in the wells can be attached to the membrane and grown thereon. The cells are fed as nutrients pass from the nutrient medium through the membrane and to the cells at a rate controlled by the concentration gradient of nutrients from the medium to the cells. The nutrient medium in the feeding tray is periodically replenished to maintain cell growth. It is desirable to effect replenishment of the nutrient medium quickly and in a manner that avoids damage to the membranes and the cells.
After the desired level of cell growth on the membranes of the wells has been attained, the multiwell filter plate can be utilized in conventional assay methods. These assay methods generally are effected by positioning the membranes and cells on the multiwell filter plate into the wells of the multiwell receiver plate, such as a 96 well receiver plate positioned below the multiwell filter plate or it just has to have the same number of wells in register with the cell/filter plate. The wells of the multiwell receiver plate contain a liquid composition to be assayed. The composition to be assayed diffuses into the cells if present and then through the membrane. The resultant liquid products within the wells of the multiwell filter plate or in the wells of the multiwell receiver plate then is assayed to determine the capability of the composition being assayed to permeate the cell barrier.
An important component in the drug discovery and development process is the determination of the oral absorption and bioavailability of new compounds. In order to perform this evaluation in a cost effective, high throughput and sensitive assay, it is ideal to use an in vitro device with a multitude of wells containing cells, a small amount of assay material and automation. Classically, the determination of in vitro oral absorption characteristics is performed using a defined epithelium cell line and measuring the apparent transport rate of the drug across a monolayer of the cells. More recently it is possible to rank/order the passive transport rate of potential drug candidates using an artificial membrane barrier. The values generated from these in vitro experiments are valuable methods for screening the most likely successful drug candidates long before the oral absorption rate are validated by in vivo measurements. A typical experiment for determining the drug absorption characteristics of a known or unknown chemical compound is performed as follows. The multiwell device is seeded with epithelium cells on top of the filter in a defined nutrients medium. The same medium is also added to the single well feeding tray, located below and in fluid contact with the device containing the cells. The cells are allowed to proliferate and differentiate over a number of days. The nutrient medium is periodically replaced with fresh medium to replenish exhausted nutrients and remove waste and dead cells. At the end of a growing time, the cells and multiwell device are gently washed with an isotonic buffer to remove protein and residual nutrient medium. At this time, the multiwell filter plate is transferred to the multiwell receiver plate and the chemicals to be assayed are introduced to either the compartment above the cell layer or below the cells and filter support in the multiwell receiver tray. The opposing chamber is filled with drug free buffer and the multiwell device is incubated for some period of time, typically at 37 degrees Centigrade with shaking. If multiple time points are desired, sampling from either compartment can be achieved without separating the device. The amount of drug/chemical that is transported across the cell barrier can be determined by a variety of analytical methods, but typically is determined using LC-MS/MS (Liquid Chromatography—Mass Spectrometry—Mass Spectrometry).
After the cells have been satisfactorily grown and the feeding tray is to be replaced by the multiwell receiver plate, it is desirable to minimize transport of the nutrient medium to the multiwell receiver plate thereby to minimize dilution of the composition being assayed. Thus, it is desirable to remove any droplets of nutrient medium retained on the lower surfaces of the membranes after the multiwell filter is removed from the nutrient medium in the feeding tray.
Accordingly, it would be desirable to provide a multiwell test apparatus which is capable of minimizing transport of nutrient medium to be mixed with a liquid composition to be assayed.